The amplification, detection and quantification of specific target nucleic acid sequences is useful in a variety of contexts, including microorganism classification, identification of genetic abnormalities including inborn errors of metabolism, diagnosis of infectious diseases, forensic analysis, environmental testing, and studies involving developmental and cellular biology.
The primary approach to polynucleotide detection involves discrimination based on sequence specificity. Typically, polynucleotide assays exploit the capacity of nucleic acids to hybridize or anneal in a sequence-specific (i.e., complementary) manner. The ability to detect polynucleotides by hybridization is dependent on the sensitivity of the assay. The sensitivity of these hybridization assays is a function of the specific activity of the probe system, comprising polynucleotides used to search for complementary sequences in a sample, along with any components used in signal generation. However, there are limits to the specific activity of a polynucleotide probe system. To further increase the sensitivity of hybridization assays, those of skill in the art have begun to increase the quantity of target nucleic acid sequence being assayed.
A variety of amplification techniques have been devised to improve the detection of nucleic acid targets by means of increasing the amount of the initial target molecule. These techniques include Strand Displacement Amplification (i.e., SDA), the Polymerase Chain Reaction (i.e., PCR), Reverse Transcription Polymerase Chain Reaction (i.e., RT-PCR), Nucleic Acid Sequence-Based Amplification (i.e., NASBA), Self-Sustained Sequence Replication (i.e., 3SR), and the Ligase Chain Reaction (i.e., LCR). Each of these techniques produces greater quantities of a target polynucleotide, thereby increasing the sensitivity of polynucleotide detection assays.
Strand Displacement Amplification (SDA) is described in Walker et al., Proc. Natl. Acad. Sci. (USA) 89:392-396 (1992), Walker et al., Nucl. Acids Res. 20(7):1691-1696 (1992), and U.S. Pat. Nos. 5,270,184, 5,422,252, 5,455,166, and 5,470,723. SDA is an isothermal amplification technique that generates DNA copies of a single- or double-stranded target polynucleotide fragment.
The original SDA methodology described in Walker et al., Proc. Natl. Acad. Sci. (USA) 89:392-396 (1992) and U.S. Pat. No. 5,455,166 discloses the amplification of the entire sequence of a single- or double-stranded target polynucleotide fragment. The technique employs two primers with each primer exhibiting a target binding region at its 3' terminus. Disposed towards the 5' terminus of each primer is a single-stranded sequence corresponding to a nickable restriction endonuclease recognition site. These recognition sequences are not necessarily complementary to any sequence in the target polynucleotide fragment, and are designed to overhang the 3' ends of an original double-stranded target polynucleotide fragment, or the 3' ends of an original single-stranded target polynucleotide and its complement. In these positions, the unbound sequences overhanging the 3' ends of the target polynucleotide and primer serve as templates for enzymatic extensions from the free 3' ends of both the primer and its target polynucleotide, respectively. Because these primers must overhang any 3' end of a target polynucleotide fragment (and the complement of the target), in order to create the required nickable site or sites (which must be double-stranded), the SDA method requires knowledge of the nucleotide sequence at both 3' ends of any double-stranded target polynucleotide fragment to be amplified. For the amplification of any single-stranded target polynucleotide, the nucleotide sequences of the 5' end and the 3' ends must be known. This is a difficult task if the sample to be analyzed contains a mixture of polynucleotide fragments from a variety of sources and in various stages of degradation or fragmentation, such as found in biological fluids, tissues, foods and water supplies. Accordingly, it is an object of the present invention to provide a method for amplification of a target nucleic acid sequence that does not require knowledge of the nucleotide sequence at the exact ends of the target polynucleotide fragment, suspected of containing the target nucleic acid sequence.
Improvements to the SDA method are described in U.S. Pat. Nos. 5,270,184 and 5,422,252. The '184 patent describes a method of amplification that involves four primers. In addition to the amplification primers (S.sub.1 and S.sub.2) that are used in the original SDA method, the method of the '184 patent includes two bumper primers (B.sub.1 and B.sub.2). To design the sequences of these four primers, the '184 method requires knowledge of the polynucleotide sequence at each end of an internal target nucleic acid sequence and, additionally, at regions flanking these terminal target sequences. These requirements present obstacles to amplification in terms of the time and effort required to sufficiently characterize the polynucleotide sequence of a targeted genomic region, such as a diagnostic portion of a pathogenic virus or a disease-linked genetic allele in humans. Additionally, the '184 method suffers from a heightened potential for artifactual and misleading results produced by spurious annealing of members of the required set of four primers.
Another improvement on the original SDA method involves "multiplex" amplification as described in the '252 and '723 patents. The method described in these patents, while allowing the simultaneous amplification of more than one target nucleic acid sequence, requires six primers (the two original SDA primers, two bumper primers, and two adapter primers) and a correspondingly greater characterization of the polynucleotide sequence of a target genome to construct the six primers than is demanded by the original SDA method or the method described in the '184 patent. Further, the greater number of primers in the reaction mixture increases the likelihood of competing amplification reactions. Accordingly, an object of the present invention is to limit the required characterization of a target genome to sequences at the termini of a target nucleic acid sequence sufficient to allow the binding of two primers under standard conditions in the art.
The Polymerase Chain Reaction (PCR) provides an alternative method for amplifying a target sequence found in a DNA molecule. Saiki et al., Science 239:487-491 (1989) and U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159. Although the PCR method only uses two primers, PCR is limited to amplifying defined regions of a target DNA and requires a thermocycling apparatus for practical implementation. Accordingly, it is an object of the present invention to provide a method that is not only capable of amplifying both target DNA and RNA, but also that does not require thermocycling or the use of thermocycling equipment.
RT-PCR is a modification of PCR that permits the amplification of a target sequence found in an RNA molecule. Myers et al., Biochemistry 30:7661-7666 (1991) and U.S. Pat. Nos. 5,310,652 and 5,407,800. In RT-PCR, the PCR cycle is preceded by the reverse transcription of an RNA target, thereby generating a cDNA product, which can then be amplified by PCR. Like PCR, RT-PCR only becomes practical with the automation of the requisite temperature changes, using a thermocycling apparatus. However, an object of the present invention is to provide a method for the amplification of a target polynucleotide that does not require thermocycling or thermocycling equipment.
Other amplification procedures that have been developed suffer from other shortcomings. Nucleic Acid Sequence Based Amplification (NASBA) and Self-Sustained Sequence Replication (3SR) will amplify RNA target molecules, producing primarily single-stranded RNA, with some single- and double-stranded DNA. NASBA is described by Kievits et al., J. Virol. Methods 35:273-286 (1991), and U.S. Pat. Nos. 5,130,238 and 5,409,818. 3SR is described by Guatelli et al., Proc. Natl. Acad. Sci. (USA) 87:1874-1878 (1990). A similar method is described in U.S. Pat. No. 5,399,491. These methods share several features, including the reverse transcription of an RNA target molecule, second-strand synthesis to yield double-stranded cDNA which contains a promoter introduced by an appropriately designed primer, and use of the promoter to generate RNA transcripts. The process relies on mesophilic microbial enzymes which are labile at elevated temperatures. This lability restricts the NASBA technique to maximum temperatures of approximately 41.degree. C. At this relatively low temperature, however, the requisite primers exhibit reduced hybridization fidelity. Consequently, the technique suffers from background problems due to the frequent introduction of the promoter sequence, from the promoter-containing primer, at unintended locations, thereby creating promoters at unwanted positions. In addition, the techniques have been found to be extraordinarily sensitive to imbalances in reaction components, particularly the levels of enzyme activities. Moreover, the predominant product is RNA, a polynucleotide that is less stable than DNA.
Another method of nucleic acid sequence amplification is the Ligase Chain Reaction (LCR), first developed by Wu et al., Genomics 4:560-569 (1989); a thermophilic version was introduced by Barany, PCR Methods and Applications 1:5-16 (1991). LCR employs two pairs of primers. In LCR, one pair of primers anneals to adjacent positions on a target nucleic acid sequence. The other pair of primers anneals to adjacent positions on the complement of the target nucleic acid sequence region that binds the first set of primers. Although the target in LCR is amplified exponentially, the method lacks fidelity because of a tendency to produce target-independent ligations. Moreover, the amplified products consist almost entirely of pre-existing primer sequences. In addition, LCR, like PCR, requires thermocycling and the thermocycling instrumentation that brings practicality to the method. Accordingly, an object of the present invention is not only to eliminate the need for thermocycling and four primers, but also to provide a method wherein the amplified product is not dominated by pre-existing primer sequences.
Therefore, a need exists in the art for a versatile, reliable, and simple method for nucleic acid sequence amplification. Such a method should be capable of amplifying both DNA and RNA and should do so exponentially, using a minimum number of primers under substantially isostatic conditions.